| Plant D-type cyclin plays an important role in cell division.we utilize the Dl-type cyclin Poptr;CYCD1;1as the research object, clone the promoter and demonstrate its characteristics, we study its expression pattern through the Agrobacterium mediated Arabidopsis thaliana flowers, we also construct the vector expression the fused protein with GFP and import it into the onion skin using the particle gun method to detect the Poptr;CYCD1;1subcellular localization. We over-express it in the Arabidopsis thaliana, tobacco and populus simonii carr. xp. nigra to research its effect, the results show as the following statements.(1) we get six transgenetic Arabidopsis thaliana lines through the homomycin screen, in the line3and in the line6, the Poptr;CYCD1;1promoter are fused into the Arabidopsis thaliana genome. GUS activity detection shows that the gene expresses in the tip of the root after germination a day, whereas, there is no signaling in the Cotyledons and hypocotyl, after germination for three days, the whole hypocotyl, root hairs and root tip are dyed. The aerial Arabidopsis vascular tissues(leaf vein and hypocotyl) indicate strong GUS signaling, though the entire root, except for the root tip shows weak fluorescence, Poptr;CYCD1;lexpression pattern is gradually increasing with the development of the vascular tissue and the aerial part is more strong than the part in the underground.(2) the Poptr;CYCD1;1open reading frame (ORF) is987bp in length, encoding a predicted polypeptide of328amino acids with a molecular weight of36735.6Da and a PI of4.97. We find the Poptr;CYCD1;1amino acid sequence has the most homology with castor-oil plant, the fluorescence of the fused protein CYCD1;1-GFP concentrates to the nuclear compared with the control, the result shows that the Poptr; CYCD1;1is localized in the nucleus.(3) We generated transgenic tobacco over-expressing Poptr; CYCD1;1gene and get12positive resistance buds, and obtain seven transgenic lines by the PCR detection, six lines through the Northern hybrid. We also acquire six Arabidopsis thaliana buds and two populus simonii carr. xp. nigra which resist the Fabio antibiotics, all of which are verified by the PCR.(4) Contrast to the same development period wild type tobacco, the T1and T2Poptr; CYCD1;1tansgenic line do not display much difference in the differentiation medium, whereas in the producing root medium, there are many differences between the wild type and transgenic ones. The transgenic ones produce root slower, thicker root and produce flower bud after10days. When the transgenic lines are moved into the soil, The transformed segregation tobacco showed more lateral branches, narrow leaves, slender and small leaf epidermis area and early flowering. The transgenic lines have bigger flowers, longer sepal and pistil, obviously bigger stigma, the transgenic lines fruit is also long and thin, shrivel, more wrinkle, thinner carpopodium, dried up placenta. The wild type seeds are satiation. So over-expression Poptr; CYCD1;1affects the tobacoo shoot, root, flowers and fruit development.(5) The wild type tobacco epidermis area is much bigger than the transgenic ones with the help of electron microscope observation. The upper and under epidermis cells, Stockade tissue, sponge tissue cell volume are all bigger than the wild type, especially the transgenic ones stockade tissues are disordered, over-express Poptr; CYCD1;1can influence the leaf structure in the tobacco.(6) Compared The transgenic Poptr;CYCD1;1with the wild type tobacco seed germination for twenty days, the wild type tobacco seeds all germinate, and the seedlings are robust. Whereas, the transgenic lines have lower germination velocity and have defect in the seed development, which can not elongate and grow normally.(7) The transgenic Arabidopsis thaliana grow more lateral roots than the wild arabidopsis, the transgenic populus grow faster with wider and curly leaves, these result reveal that the Poptr;CYCD1;1gene affects the arabidopsis root and populus leaf develppment. |
PDF Full Text Request